Rubber is found in more than two thousand plant species, but is limited to only a few plant families (See: Backhaus, Israel J. Bot., 34:283-293 (1985); and Archer et al., Chemistry and Physics of Rubber-like Substances, Bateman, L. ed., pp 41-72, MacLaren, London (1963)). Rubber is a polymer composed of between 320-35,000 isoprene molecules. These are linked by stepwise, head-to-tail, cis-1,4 condensations that form the polyisoprene chains. The stepwise isoprene additions are performed by a prenyltransferase enzyme [E.C. 2.5.1.20] known as rubber transferase (RUT), rubber polymerase and rubber cis, 1-4 polyprenyl transferase. RuT has been ascribed as the sole enzyme responsible for rubber formation in plants (See: Backhaus, Israel J. Bot. 34: 283-293, (1985); Berndt, U.S. Government Res. Rep. AD-601, 729, (1963), Archer and Cockbain, Methods in Enzymology, 15:476-480, (1969) Archer and Audley, Advances in Enzymology, 29:221-257, (1967) and Lynen, Rubber Res. Inst. Malaya, 21:(4) 389-406, (1969)). However, as this patent application will show, there is another enzyme, known as RPP (for rubber particle protein) that clearly causes rubber biosynthesis when transferred to a foreign host plant, which in our case, is tobacco. As this patent application will show, RPP, unlike RuT, is not a prenyltransferase, but an unusual form of allene oxide synthase (AOS) which is a member of a class of cytochrome P450, heme-binding enzymes. We will show that RPP has properties similar to other members of this class of heme-binding enzymes and that it is an enzyme responsible for rubber biosynthesis. P450 enzymes have never before been implicated in rubber biosynthesis. We will describe the construction of a binary vector, named pBIRPP, which contains the RPP gene and will show that when the RPP gene is transferred to tobacco and expressed, it produces functional RPP enzyme. The resulting RPP enzyme produced in these transgenic plants causes rubber biosynthesis and the rubber so produced accumulates in rubber particles in the cells of these plants. We predict that guayule RPP and its homologues from other rubber-producing species are the enzymes responsible for rubber biosynthesis in plants.
The enzymatic activity of RPP has never been elucidated until now. Spectral analysis will be described which confirms that RPP is a cytochrome P450. It will be shown that RPP is a non-mono-oxygenase type of heme-binding protein and is not a prenyltransferase. Knowledge of such a role for P450 in rubber has never before been described. Biochemical analyses will show that RPP metabolizes lipid hydroperoxides into lipid epoxides and does not perform prenyl-transferase-type reactions. This patent application goes on to describe the means and methods for isolating a full-length cDNA for the guayule RPP gene. It describes the DNA and amino acid sequence for RPP and demonstrates a method for inserting the RPP cDNA downstream of a strong eukaryotic CaMV 35S plant promoter in the pBIRRP binary vector. It describes how this vector is used to create transgenic tobacco plants which then produce functional RPP. These transgenic tobacco plants express the foreign RPP gene contained in the pBIRPP vector and carry out rubber biosynthesis. Rubber is observed as rubber particles which accumulate in tobacco cells. These particles resemble those observed in guayule cells. The invention verifies that RPP, encoded by a single nuclear gene, is responsible for rubber biosynthesis in plants. Moreover, it suggests that the mechanism of rubber formation is not based solely on the presence a RuT-type, cis-prenyl-transferase enzyme, as originally thought, but also requires a RPP-type, cytochrome P450, heme-binding enzyme.
The first identification of guayule RPP was made in 1985 (Backhaus and Chandra, in Alcorn, S. and Fangmeier, D. (eds) Proceedings of the 4th International Guayule Conference on Guayule Research and Development, Oct. 16-19, (1985); Backhaus and Bess, in Randall, D. D. et al. (eds) Current Topics in Plant Biochemistry and Physiology, Vol 5:186 (1986)). Subsequent publications described its purification and characterization and suggested a putative role as a prenyltransferase in guayule rubber particles (Backhaus and Bess, in Benedict, C. R. (ed) Biochemistry and Regulation of cis-Polyisoprene in Plants, NSF sponsored workshop publication, p. 204-220 (1986); Cornish and Backhaus, Phytochem. 29:3809-3813, (1990); and Backhaus et al. Phytochem. 30:2493-2497 (1991)). Work by Cornish and Siler demonstrated that RPP-like proteins were also present in Hevea and Ficus (Siler and Cornish, Phytochem. 32: 1097-1102 (1993); Cornish, et al. J. Nat. Rubb. Res. (1994) (in press); Cornish, et al., Phytochem. (in press) (1994); Siler and Cornish, Phytochem. (1994)(in press)). The RPP-like proteins in rubber particles of these other species are likely involved in the same enzymatic reaction as RPP. These are the only known citations in the literature that specifically refer to RPP. Reference to another protein isolated from guayule rubber particles was made (Benedict et al. Plant Physiol. 92:816-821 (1990)) which described a prenyl-transferase but did not identify that protein as RPP despite prior knowledge by those authors (cf. Benedict, C. R. (ed) Biochemistry and Regulation of cis-Poly-isoprene in Plants, NSF sponsored workshop publication, p. 204-220 (1986)) of RPP's existence in guayule rubber particles. The first identification and verification of RPP as a cytochrome P450 necessary for rubber biosynthesis is this patent application.
The molecular cloning of RPP is described herein. Prior to this, its molecular weight was estimated to be 48,500 to 53,000 Daltons as determined by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Other characteristics of RPP includes its amino acid composition, its isoelectric point (pI=6.2), that it is a glycoprotein and how it is assembled in rubber particles as a membrane protein (Backhaus et al., Phytochem. 30:2493-2497 (1991)). In this patent application we further describe its amino acid sequence, its biochemical function and the consequences of its expression in tobacco, a non-rubber producing species.
RPP is the most abundant protein of guayule rubber particles (Backhaus et al., Phytochem. 30: 2493-2497 (1991)) and was, therefore, considered to be important for rubber biosynthesis. For this reason, the cDNA for the RPP gene was targeted for isolation from guayule stembark, with the intent of using the gene to induce rubber biosynthesis in a variety of other organisms that do not ordinarily synthesize rubber. This invention teaches that this occurs when the RPP gene is placed in a binary plasmid vector under the control of a strong promoter and is transferred to a new host (tobacco) which expresses the RPP gene and produces functional RPP enzyme. Conversely, the gene can also be placed in other plants, such as guayule or Hevea, that already synthesize rubber with the intent of overexpressing RPP. The overexpressed RPP can then enhance rubber production in these plants. Additionally, RPP could be transferred to and expressed in other eukaryotic organisms or cell cultures including yeast, insects or higher animals or in prokaryotic organisms, with the intent of producing rubber. This patent application seeks to protect the invention of the isolated DNA molecule for the RPP gene from guayule and demonstrates that this gene can generate rubber biosynthesis in those transgenic organisms (ie., tobacco) when that foreign RPP gene is expressed.